Protein crosslinking reagents cleavable within acidified intracellular vesicles

ABSTRACT

Crosslinking reagents for amino group-containing compounds are provided, which crosslinkers can be cleaved under mildly acidic conditions. The crosslinkers can be used to crosslink biologically active substances to be delivered to the cells, wherein the crosslinker will be cleaved in the mildly acidic conditions within the cell.

FIELD OF THE INVENTION

The present invention relates to protein crosslinking reagents which canbe cleaved under acidic conditions. The crosslinked products so formedcan be used to deposit within a cell a non-crosslinked protein or othermolecule which is transported to the cell as a crosslinked proteinutilizing receptor-mediated endocytosis; to construct therapeuticprodrugs; and to reversibly couple proteins to matrices for syntheticand chromatographic purposes.

BACKGROUND OF THE INVENTION

There are numerous situations wherein it may be desirable to control therelease of amino-group-containing substances to liquid media. Forexample, it may be desirable to control the release of anamino-group-containing drug or cytotoxin to a cell population orspecific members of a cell population. It may also be desirable tocontrol the cleavage of various cross-linked proteins or peptides, forexample, in analyzing the spatial relationships in a complex of largeamino-group-containing molecules such as peptides or proteins.

One specific situation in which controlled release is desirable is indelivering a biologically active compound through the cell membrane toinner cell structures, for example, where the compound has a low orreduced effect if trapped in the medium outside the cell membrane but ismore potent once released inside the cell.

It is also desirable to be able to deliver biologically active compoundsto selected cells in a heterogeneous cell population. For example, intreating diseased or infected cells such as virus-infected cells ortransformed or malignant cells, it is desirable to deliver cytotoxinsanti-viral agents or growth regulating factors to the diseased ormalignant cells but not to normal cells

One approach disclosed for targeting biologically active compounds tomalignant cells uses an antibody-toxin conjugate. The antibody isspecific for malignant cells and delivers the toxin to them. To beeffective, these systems should deliver the toxin with high selectivityto the target cells without unnecessarily reducing the effectiveness ofthe active substance. These problems are particularly important wherethe goal is destruction of infected or diseased cells in vivo withoutharming normal cells.

A variety of protein crosslinking reagents are commercially available.Those crosslinkers in widest use are heterobifunctional reagents usingmaleimide and N-hydroxysuccinimide esters. Specific coupling of twoproteins via a non-cleavable thioether bond is achieved by introducingan SH group into one of the proteins, as disclosed by Kitagawa, U.S.Pat. No. 4,150,033. Commercially available cleavable crosslinkers arelimited to disulfide, carboxylic acid ester, and vic-glycol groupingsrequiring -SH reagents, strong nucleophiles, or periodate oxidation,which permanently modify many proteins, cf. Wang et al., Isr. J. Chem.12:375-378, 1974; Lutter et al , FEBS 48 288-292, 1974.Disulfidereagents are only slowly cleaved within the cells.

It was found by Neville, CRC Crit. Revs. Ther. Drug Carrier Syst.2:329-352, 1986, that the efficacy of holo-toxin conjugates coupled viadisulfide bonds is not superior to thioether crosslinking.

Additional cleavable bifunctional crosslinking reagents are known,including Lambert et al., J. Mol. Biol. 149: 451-476 (198I) and Wang etal., Isr. J. Chem. 12: 375-389 (I974) wherein are disclosed bifunctionalcrosslinking reagents containing a cleavable disulfide bond The reagentsare used to characterize biochemical systems.

Carlsson et al., Biochem. J. 173: 723-737 (1978) disclose a procedurefor forming disulfide bonds between two different proteins using thebifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate.

More specifically, certain monoclonal antibodies, toxins, and conjugatesthereof are known.

Vitetta et al, in Science 219: 644-650 (1983) and Edwards, Pharmacol.Ther. 23: 147-177 (1983) disclose disulfide-linked conjugates of toxinsand monoclonal antibodies specific to cell-surface structures. Theseconjugates are used to target toxins toward specific cells havingsurface structures recognized by the antibodies.

Ramakrishnan et al., in Cancer Research 44: 1398-1404 (1984) discloseconjugating pokeweed anti-viral protein (PAP) to anti-Thy 1.1, amonoclonal antibody. The conjugate is used to inhibit protein synthesisselectively in Thy 1.1-positive target leukemia cells. The linker usedto form the conjugate is N-succinimidyl-3-(2-pyridyldithio)propionate.When the disulfide bond is cleaved, the free PAP toxin is produced.

Ritz et al., Nature 283: 583-585 (1980) disclose a monoclonal antibody(J5) that is specific for common acute lymphoblastic leukemia antigen.

Stirpe et al. , J. Biol Chem. 255: 6947-6953 (1980) disclose a methodfor preparing gelonin, a protein cytotoxin.

Barbieri et al., Biochem. J. 203: 55-59 (1982) disclose the purificationand partial characterization of an antiviral protein known as pokeweedantiviral proteins ("PAP-S").

Neville et al., U.S. Pat. No. 4,359,457, disclose a conjugate ofanti-Thy 1.2 monoclonal antibody and ricin used as a tumor suppressivecomposition against lymphoma. The linking agent used ism-maleimidobenzoyl-N-hydroxysuccinimide.

The above-described approaches either depend on the toxicity of anantibody-toxin conjugate, or they depend on disulfide bond cleavage, aphenomenon that may be difficult to control temporally and spatially toavoid release of the toxin before delivery to the targeted cells.

An acid cleavable protein crosslinker has been described based on acitraconic anhydride group by Blattler et al., U.S. Pat. Nos. 4,542,225,4,618,492. Deficiencies in this scheme include the fact that thehydrolysis rate is not linearly correlated with the hydrogen ionconcentration (a 30-fold change in hydrolysis for a 600-fold change in[H+], there is a potential for forming an irreversible crosslink throughMichael type additions of cellular SH groups to the double bond, thereis a failure to demonstrate significant hydrolysis of crosslinkedproduct at the low end of the intravesicle pH (5.4), i.e., <25%hydrolysis at pH 5.5 in 10 hours, and there is a failure to demonstrateefficacy over non-cleavable crosslinkers in a system involving proteinuptake either by tissue culture or in vivo, as well as a lengthysynthetic sequence.

Kirby et al. , Proc. Biochem. Soc. Symp. 31: 99-103 (1970) disclose thatmaleic acid amides are rapidly hydrolyzed below pH 3, and thatsubstitution of maleamic acid increases that rate, with a t-butylsubstituent providing the largest increase and a methyl substituent thesmallest.

Dixon et al. , Biochem. J. 109: 312-314 (1968) disclose reversibleblocking of amino groups using 2-methyl-maleic (citraconic) anhydride asa blocking agent. The amine bond between the citraconyl residue and alysine residue of insulin was not cleaved at pH 6.5; when the pH waslowered to 3.5 at 20° C. overnight, there was total release of theblocking group, leaving the insulin unchanged.

The concept of a prodrug is not new, and has been described by Albert inNature 182:421-423, 1975. The acid catalyzed hydrolytic properties oforthoesters, acetals, and ketals are well described, particularly byCordes et al in Chem. Rev. 74 581-603, 1974, and have been used toachieve prodrugs in the slow release of subcutaneously implanted steroidcontraceptives from a solid orthoester polymer matrix, Heller et al. ,Polym. Eng. Sci. 21: 727, 1981, and in the gastric release ofacetaminophen from a more pleasant tasting acetal prodrug, Hussain, U.S.Pat. No. 3,786,090. The log of the hydrolytic rate constant of this drugwas found to be linear with pH over the range of pH 2-6, Hussain et al.,J. Pharm. Sci. 67: 546-546 1978.

The extension of the above basic chemistry to a protein crosslinkercapable of release within intracellular compartments was not trivial.The starting materials for orthoester synthesis, ketene acetals, arenotoriously difficult to work with because of cationic polymerizationside reactions. Synthetic routes could not use strong acid conditions,and the desired maleimide functionality limited many approaches, such asorthoesters via the Pinner synthesis. These limitations wereparticularly bothersome for the synthesis of the heterobifunctionalreagents which classically employ harsh conditions

Neville et al., in U.S. Pat. Nos. 4,356,117; 4,359,457; 4,440,747;4,500,637; 4,520,011; and 4,520,26; elucidate the concepts and utilityof constructing monoclonal antibody-protein toxin conjugates(immunotoxins) directed at specific unwanted target cells. Holo-ricinbased immunotoxins have greater efficacy than ricin A chainimmunotoxins. The enhanced efficiency of such holotoxin conjugates overthose constructed with toxin A-chains has been documented. Thediscrimination between target and nontarget cell was maintained byreversibly blocking the ricin toxin B chain binding site with lactose,since this site has been shown to be essential for full immunotoxinefficacy, as lactose is extruded from the cell following endocytosis.The ricin binding site is required for efficient membrane translocationof ricin within the cell, and lactose blocks ricin binding outside thecell but not inside the cell because lactose is actively transported outof the cell. However, such conjugates reversibly blocked with lactosehave limited in vivo efficacy, since high concentrations of lactosecannot be maintained in vivo without untoward effects.

Acid cleavable crosslinkers permit reversible blockade of the ricinbinding sites by crosslinking asialoglycoproteins, andasialoglycopeptides, and mannose binding proteins to these sites whichcan dissociate in the acidified vesicle. In addition, the translocationfunctions of mutant diphtheria toxins such as CRM 103 and CRM 107, whichare partially blocked by coupling to monoclonal antibodies withconventional crosslinkers (Greenfield et al. Science 238 536-539, 1987)can be uncoupled within the cell using a cleavable crosslinker. Sincethese toxin mutants, which are binding domain mutants, have reducednon-target cell specificity to begin with, they should be suitable forin vivo use Immunotoxins of CRM 103 and CRM 107 constructed with acidcleavable crosslinkers should provide an extra I to 2 logs increase inefficacy over the conventional crosslinkers.

Polyethylene glycol (PEG) has been conjugated to proteins by a varietyof procedures to block certain functional domains in vivo, Aubuchowskiet al., J. Biol. Chem. 252: 3582-3586, 1976, These PEG conjugates can beused to administer an enzyme protein missing from the body in order tocorrect an enzyme deficiency disease. PEG coupling can minimize twoproblems, namely, rapid clearance of the unmodified protein from thevascular system, either antibody or extra antibody mediated, and theformation of antibodies to the foreign protein. Rapid clearance andantigenic stimulation are also problems concerning the in vivo use ofimmunotoxins. However, PEG, like antibody coupling, also interferes withtoxin translocation.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the above-mentioneddeficiencies in the prior art.

It is another object of the present invention to provide crosslinkingreagents for proteins which can be cleaved under mild acidic conditions.

It is still another object of the present invention to provide a methodfor synthesizing crosslinking agents.

It is a further object of the present invention to provide a method todeposit efficiently within a cell a non-crosslinked protein or othermolecule which has been previously transported into the cell as acrosslinked protein or other molecule, using receptor-mediatedendocytosis.

It is yet another object of the present invention to provide therapeuticprodrugs.

It is yet a further object of the present invention to reversibly coupleproteins to matrices for synthetic and chromatographic purposes.

It is still a further object of the present invention to providecompositions which can be used in the treatment of cancer.

It is still another object of the present invention to providecompositions which can be used in the treatment of acquired immunedeficiency syndrome (AIDS).

It is still a further object of the present invention to providecompositions which can break the infectious cycles of viruses.

Yet another object of the present invention is to provide compositionswhich can be used to kill T cells.

According to the present invention, compounds are provided which permitthe crosslinking of two amino and/or sulfhydryl group containingsubstances at pH >7, and which will release these substances undermildly acidic conditions with little or no attenuation of activity.

The preferred biologically active substances to be delivered to thecells by the method of the present invention is a protein or a peptideor a drug or an enzyme or a nucleic acid. Most preferably, the activesubstance is a cell toxin to be delivered to selected cells, such asricin, diphtheria toxin, abrin, and other ribosomal-inactivatingproteins or elongation factor 2 inactivating proteins Peptide toxins arepreferred because they are readily bound to the reagent and because theyare extremely potent toxins which inhibit cell protein synthesis whenpresent inside the cell in extremely minute quantities. Otheramino-group-containing cytotoxins which are not peptides are also withinthe scope of the invention, such as melphalan, bleomycin, adriamycin,and daunomycin.

The conjugates are delivered to selected cells by binding partners tocell-surface features. The preferred binding partners are antibodies tocell surface antigens Particularly preferred are monoclonal antibodiesto cell surface antigens specific to diseased, infected, transformed, ormalignant cells, but not to normal cells. Particularly, but notexclusively, they are antibodies that are taken up by the cells. It isnot necessary that non-target cells lack the specific antigen entirely,as long as the antigen is not present in sufficient numbers on thosecells to permit significant uptake of the active substance by the cells.

Examples of such antibodies are antibodies to melanoma surface antigensand the antibodies to surface antigens found on T-cells and T-celllymphomas, such as CD-3, CD-4, CD-5, CD-11, and CD-12.

Other binding partners that can be used include non-antibody cellmembrane transport agents such as transferrin and protein hormones orgrowth factors such as insulin.

The hydrolytic rates are such that these crosslinkers are cleaved withinminutes or hours at the pH of acidified cellular vesicles, pH 5.4, yetare 100 times more stable at the intravascular pH of 7.4, and 1000 timesmore stable at a storage pH of 8.4.

The essential points of the present invention are that the cellularcomponent causing cleavage is not appreciably present in the serum, thecellular component causing cleavage is present within a compartment towhich the immunotoxin is routed by the targeting moiety, and theintracellular cleavage is sufficiently rapid to restore substantiallyfull activity of the active molecule.

The crosslinking agents of the present invention comprise the unit:##STR1##

wherein A is bridge unit which is unreactive with the protein or othermolecules to be crosslinked and has functionalities which are compatiblewith maleimide groups. The bridge may include peptide chains, aromaticsubstituents, and the like. A is preferably (CH₂)_(n), where n is aninteger of from 1 to 8. Methyl groups (CH₃) are the preferred centralcarbon substituent but other groups having similar electron donatingcapacity such as C₂ -C₉ alkyl, phenyl, or substituted phenyl, can beused.

In a second aspect of the invention, a heterobifunctional acid cleavablecrosslinker is provided which is suitable for linking an amino groupcontaining substance to a sulfhydryl group on a second compound. Thisreagent has the general formula: ##STR2## wherein A and B are bridgeunits, A being the same as defined above, and B is: ##STR3## where n isan integer of at least 1.

In a third aspect of the present invention, methods are provided forsynthesizing the crosslinking reagents of the present invention eitherby ketal exchange reactions between commercially available ketals andalcohols containing a maleimide functionality, or the acid catalyzedaddition of phenols derivatized with an N-hydroxysuccinimide estergrouping to vinyl ethers bearing a maleimide moiety.

In a fourth aspect of the present invention, homobifunctionalcrosslinkers are provided in which the acid labile moiety is anorthoester function and which contain the maleimide group for reactionwith sulfhydryl containing compounds. These crosslinkers have thegeneral structure: ##STR4## wherein A is a bridge unit as defined above.

The fifth aspect of the present invention features a method for thesynthesis of the orthoester class of crosslinkers by the reaction of anappropriate alcohol with a bis-ketene acetal. FIGS. 1, 2, and 3illustrate in flow diagram form the synthesis of ketal, acetal, and theorthoester class of crosslinkers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of crosslinker 1.

FIG. 2 shows the synthesis of heterobifunctional crosslinker 2.

FIG. 3 shows the synthesis of orthoester crosslinkers 3 and 4.

FIG. 4 shows the fractional loss of DT dimers crosslinked withcrosslinker 3 (circles) and crosslinker 4 (crosses) as observed withtime following acidification to pH 6.4. The loss of dimers is correlatedwith the appearance of monomers. The reaction kinetics is first order.

FIG. 5 shows the fractional loss of the immunotoxin T101-DT plotted on alog scale, followed by the appearance of free T101 antibody at pH 3.5,4.5, and 5.5. The slope at each pH defines the observed first order rateconstant of dissociation, k, and in the insert, log k is plotted vs pH,revealing a linear dependence, a characteristic of specific acidcatalysis.

FIG. 6 shows that the increasing concentration of DT progressivelyinhibits the protein synthesis of Vero cells. Dimers crosslinked bycrosslinker 3 are less toxic, requiring 30-fold higher concentrationsfor equal inhibition. Treatment at pH 6.5 of the crosslinked dimer fortwo hours converts about half the dimer into monomer with partialrestoration of the original toxicity.

FIG. 7 shows the potency of DT and T101 (Anti-CD5) conjugates of DT,assayed by protein synthesis inhibition, made with the non-cleavablecrosslinker BMH and the cleavable ketal crosslinker 1, compared onnon-target (Vero) cells (panel b, right) lacking CD5 surface antigen andtarget (Jurkat) cells (panel a, left) carrying CD5. The ketal linkedconjugate is 50-fold more potent than the BMH conjugate on target cells.Jurkat assay, 6 hours at 37° C.

FIG. 8 shows the potency of ricin and T101 (Anti-CD5) conjugates ofricin assayed by protein synthesis inhibition made with thenon-cleavable crosslinker BMH and the cleavable ketal crosslinker 1,compared on non-target (Vero) cells (panel b, right) lacking CD5 surfaceantigen and target (Jurkat) cells (panel a, left) carrying CD5. Theketal linked conjugate is 10-fold more potent on the target cells. Thedashed lines are plus 100 mM lactose.

FIG. 9 shows a comparison of the ricin binding site of an Anti-CD5(T101) immunotoxin reversibly blocked by the inclusion of asialofetuinlinked to ricin via crosslinker; 1 A similar but non-reversibly blockedimmunotoxin was constructed with BMH.

FIG. 10 shows intracellular hydrolysis of 35S-cysteine from theconjugate transferrin-crosslinker 1.

FIG. 11 illustrates the application of crosslinker 1 in reversiblycoupling a protein to a matrix.

DETAILED DESCRIPTION OF THE INVENTION

The cleavable crosslinkers of the present invention have three majorutilities:

1. The crosslinkers can be used to efficiently deposit within a cell anon-crosslinked protein or other macromolecule which is transported intothe cell as a crosslinked protein utilizing receptor-mediatedendocytosis dictated by one of the crosslinked proteins. An example ofthis is the efficient loading of ricin and diphtheria toxin into cellsvia an anti-CD5 monoclonal antibody-toxin complex (immunotoxin), whichcan be used to kill T cells. Immunotoxins constructed with thesecleavable crosslinkers are 10-50 times more toxic toward target cellsthan the best conventional non-cleavable crosslinkers.

2. The crosslinkers of the present invention can be used to constructtherapeutic prodrugs. A specific functionality of an administeredtherapeutic protein or an agent attached to a protein is blocked whileoutside of the target cell, but is unblocked within the target cell.This achieves a higher therapeutic ratio by reversibly alteringfunctionalities which direct proteins to certain cells or organs.Lifetime within the vascular compartment, clearance by preexistingantibodies, and antigenic stimulation can also be modified in this way.One example of the reversible blocking of a protein functionality is theblocking and unblocking of protein toxin binding sites which iscorrelated with cellular toxicity.

3. Crosslinkers cleavable under non-denaturing conditions are valuableresearch tools to reversibly couple proteins and other compounds havingamino and/or sulfhydryl groups to matrices for synthetic andchromatographic purposes, to assess neighbor-neighbor interactions, tofollow the passage of macromolecules through intracellular compartments,some of which are acidified, and to assess the inhibitory effects ofmolecular derivatization procedures.

All of the crosslinkers of the present invention, with the exception ofcompound 2, are homobifunctional reagents containing the maleimidefunctionality. Maleimide groups are highly specific for sulfhydrylgroups which can be readily generated on a protein or other amino groupcontaining substance by methods well known in the art. Theheterobifunctional reagent 2 forms a crosslink between an amino group onone component and a sulfhydryl group on another component.

With respect to the present invention, the nature of the crosslinkerjoining the two protein molecules is well defined. The crosslinkershould be easy to synthesize in large amounts, and should have goodstability for long-term storage. The coupling reaction should take placeunder mild conditions which do not affect protein structure or activity.The crosslinked product should be reasonably stable during storage andshould not be cleaved prematurely, i.e., the linkage should be inert atpH at least 7.4, but should be rapidly cleaved in the pH range or 5 to6, releasing the individual proteins without attenuation of activity

Synthesis of a Bis-maleimide Ketal Crosslinker (FIG. 1)

This synthesis involves a ketal exchange reaction betweenN-(2-hydroxyethyl) maleimide and 2,2-dimethoxypropane. The exchange iscatalyzed by trace amounts of acids, e.g., p-toluenesulfonic acid.

N-(2-hydroxyethyl) maleimide was prepared according to the method ofKosower et al., J. Med. Chem. 14: 873-838, 1971. 141 mg of this materialwas suspended in 10 mL benzene, and 75 mg of 2,2-dimethoxypropane wasadded. The mixture was stirred at room temperature under an argonatmosphere and 0.25-0.5 mg p-toluenesulfonic acid was added as acatalyst. After the reaction mixture turned homogeneous, the solvent wasdistilled off and another 50 mg portion of 2,2-dimethoxypropane in 10 mLbenzene was added. Stirring was continued at room temperature for anadditional two hours. A few drops of pyridine were added to neutralizethe acid. The solvents were distilled off, and 10 ml of benzene wasadded to the residue. The benzene soluble portion was decanted off andevaporated to dryness. The residue was crystallized from acetone-hexaneto yield white, fluffy crystals, mp 124°-126°.

Crosslinker 5, similar to crosslinker 1 with one less CH₂ group in eachbridge was prepared in a similar manner and isolated as a crystallinesolid.

Synthesis of a Heterobifunctional Acetal Crosslinker (FIG. 2)

This synthesis comprises adding a phenol derivatized with aN-hydroxysuccinimide ester functionality to a vinyl ether containing amaleimide group. The addition is acid catalyzed.

Preparation of 2-Maleimidoethyl vinyl ether

A quantity of 1.18 g of N-(2-hydroxyethyl)maleimide was suspended in 25ml ethyl vinyl ether and refluxed with stirring under an argonatmosphere, 70 mg mercuric acetate was added, and the reaction wascontinued at reflux for 24 hours. After 24 hours, the reaction mixturewas allowed to cool to room temperature, and 1.50 mg of anhydrouspotassium carbonate was added. After stirring for a few more minutes,the mixture was filtered and the precipitate was washed thoroughly withethyl acetate. The washings were combined with the filtrate andevaporated to dryness in vacuo to yield an oily residue. This oilyresidue was chromatographed over 50 grams of neutral alumina, activityIII, using methylene chloride-hexane (1:1) for elution. The initialfractions were collected and combined to yield 200 mg of crude2-maleimidoethyl vinyl ether. The product was suitable for use in thenext step in preparation of the crosslinker, but for analytical purposescould be purified by rechromatography over alumina or kugelrohrdistillation.

Crosslinking Reagent

One hundred twenty mg of 3-(4-hydroxyphenyl)propionicacid-N-hydroxysuccinimide ester was dissolved in 10 mL of anhydrousethyl acetate, and 200 mg of maleimidoethyl vinyl ether was added. Thereaction mixture was stirred at room temperature under argon, and asolution of 0.5 mg of p-toluenesulfonic acid in 1 mL anhydrous ethylacetate was added as a catalyst. After a reaction time of six hours, afew drops of pyridine were added to neutralize the acid, and thereaction mixture was evaporated to dryness to yield a viscous paleyellow oil. Hexane was added to this residue, and the mixture wasrefrigerated for several hours. The white solid which precipitated, 200mg, was collected and washed with hexane. Crystallization from methylenechloride-hexane yielded white crystals, mp 134°-147°.

Synthesis of Orthoester Crosslinkers, FIG. 3

These crosslinkers are prepared by the addition of the appropriatealcohols ROH to the bisketene acetal ##STR5## to give the orthoester ofthe general formula ##STR6## wherein R is C₁ -C₉ alkyl.

The ketene acetal was prepared from the spiro compound ##STR7## by arearrangement induced by hexane solution of n-butyl lithium andethylenediamine according to the procedure of Heller et al., 1986,submitted to Macromolecular Synthesis.

Two hundred fifty mg of N-hydroxymethyl maleimide, prepared frommaleimide according to the method of Tawney et al. J. Org. Chem. 26:15-21, 1961, was dissolved in 50 mL anhydrous ether, and the solutionwas treated with 220 mg of the ketene acetal. The homogeneous reactionmixture was stirred under an argon atmosphere at room temperature forone to two hours. A slight turbidity was observed, probably as a resultof polymerization of a portion of the ketene acetal. The reactionmixture was filtered and concentrated to remove most but not all of thesolvent. Hexane was added dropwise to initiate crystallization. Themixture was cooled in a refrigerator to complete crystallization. Thewhite crystals were isolated by filtration and dried to yield 350 mg ofthe orthoester 4, mp 143°-146° (d).

An orthoester crosslinker 3 was prepared in the same manner, usingN-hydroxyethyl maleimide in place of N-hydroxymethyl maleimide. Thecrosslinker in this example formed a colorless oil.

Other examples of acid cleavable crosslinkers are as follows:

Heterobifunctional acid cleavable ketal crosslinkers of the formula##STR8## wherein n≧1 ##STR9## wherein A is bridge unit as described in p10. Methyl groups (CH₃) are the preferred central carbon substituent butother groups having similar electron donating capacity such as C₂ -C₉alkyl, phenyl, or other substituted phenyl, can be used. ##STR10##wherein A is as defined above.

Heterobifunctional acid cleavable acetal crosslinkers of the formula##STR11## wherein A is as defined above.

Homobifunctional acid cleavable orthosester crosslinkers of the formula##STR12## wherein R is alkyl of C₁ -C₈ and n≧1. ##STR13## wherein R isalkyl of C₁ -C₈ and n≧1. ##STR14## wherein n≧1, R' is methyl or higheralkyl (C₁ -C₈) and R is hydrogen or alkyl of C₁ -C₈.

Advantages of Multiple Crosslinking Units

From the insert in FIG. 5, it can be seen that the slope of log K_(obs)vs pH is 1. It would be advantageous to have K_(obs) =k'[H]^(x) wherethe exponent x is at least 2. This would provide a 10,000-fold change orgreater in the hydrolytic rate for a 100-fold change in [H⁺ ] betweenthe vascular compartment and the endosomal compartment. This can beachieved by having the order of the reaction at least 2 with respect to[H⁺ ]. A typical chemical example is the dissociation of heavy metalsfrom tetra acetate-acetic acid complexes. Using the present chemistry,two crosslinking units will provide a dependency on K_(obs) =k'[H]^(x)where x approaches 2 at low values of the fraction of hydrolyzedmaterial. The derivation comes from target theory and utilizes thePoisson formula, which predicts that, if the average number of randomevents in a single target is a, then the fraction of targets havingexactly n events is ##EQU1## and the fraction of targets surviving, P,this is when n =0 is equal to e^(-a).

This analysis can be extended to a multitarget model containing ntargets (Hutchinson and Pollard in Mechanisms in Radiobiology Vol 1,General Principles [Errera and Forssbert, eds.] 1961, Academic Press,New York and London.) For two targets P=e^(-a) (1+a) and for threetargets, ##EQU2## The result for multi hit targets is that at low valuesof a, which may be equated to the product of an acid independent firstorder hydrolytic rate constant k', [H⁺ ] and time, P is increased. Forexample, for n=0, P=e^(-a). At a=1. P=0.37. By going 100-fold higher inpH (time held constant), P increases to 0.99.

For two crosslinkers in parallel, the two hit model requires a =2.2 forP =0.37. However, for a=0.022 (100-fold change), P is now 0.99976, asignificant increase. In practical terms, multiple crosslinks ofsterically hindered toxin binding site are achieved in three ways:

1) Forming MAb-toxin conjugates with cleavable linkers in which thecomplex has the formula MAb-X-toxin-X-MAb. In practical terms, this isaccomplished by doubling the thiol derivatization of the toxin two-fold,lowering the thiolation of the MAb by 0.5, and inputting reactants at aratio of MAb to toxin to 1:1 to 4:1.

2) Synthesizing crosslinkers containing parallel cleavable bonds (X)placed between the protein coupling units Y and Z where B are bridgingunits, preferably C₁ -C₈ alkyl, ##STR15##

3) Using steric blocking groups such as polyethylene glycol wheremultiple polyethylene glycol units are attached to the toxin through thecleavable crosslinkers X

MAb - X-Toxin-(X-PEG)n wherein n is greater than 2.

Another group of crosslinkers according to the present invention areacid reversible crosslinkers containing an azide group forphotoinitiated crosslinking. A specific example of this type ofcrosslinker is ##STR16##

The acid cleavable crosslinkers of the present invention can be used toreversibly couple proteins to matrices. These immobilized proteins canthen be synthetically manipulated with ease, and finally released fromthe matrix by adjusting the pH. FIG. 11 is a specific illustration ofsuch a use.

Crosslinking of Proteins with Acid Cleavable Reagents Crosslinking withCleavable Heterobifunctional Reagents

The reaction conditions for crosslinking with cleavableheterobifunctional reagents are similar to those for use with thenon-cleavable linker m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)except that the coupling appears to be more efficient with crosslinker2.

Synthesis of Anti-CD5-diphtheria toxin and Anti-CD5-ricin

CD5 is human T-cell surface membrane epitope which is characteristic ofpan T cells. Both toxins, diphtheria toxin (DT) and ricin, arederivatized with crosslinker 2 using 1 mol of crosslinker 2 per mol oftoxin dissolved in dry dimethylformamide and added at pH from 8 to 8.5.Unreacted reagent is removed by passage over a small column of G25-F(Pharmacia). The maleimide derivatized toxin is reacted with thiolatedantibody (2:1 toxin:antibody) achieved by treatment of 15 mg/ml T101with 0.6 mM iminothiolane of pH 8.0, for thirty minutes, and subsequentpassage over G25-F. Both proteins are allowed to react for twentyminutes. The reactants and products are separated by HPLC chromatographyover Zorbax (Z)-250 columns from DuPont run in 90 mM Na₂ SO₄, 10 mM NaPi(a mixture of mono-and dibasic sodium phosphate), pH 8.5, 1 mM EDTA(90/10/1 buffer, 90 mM Na₂ SO₄, 10 mM sodium phosphate pH 8.5, 1 mMEDTA). The yields vary between 20 and 50% of input antibody. Allthiolations were performed under argon in the presence of 1 mM EDTA toinhibit disulfide bond formation. This procedure can be used toconjugate any IgG monoclonal antibody.

Crosslinking with Acid Cleavable Homobifunctional Reagents ProteinsWhich Form Natural Aggregates

Diphtheria toxin dimer was isolated by fractionation of previouslyfrozen diphtheria toxin in 10 mM NaPi of pH 7.1 using a 60×2.5 cm TSKcolumn (LKB). The dimer was thiolated using 1 mM of iminothiolane, pH8.0, 0.4 M Na borate, for twenty minutes. The dimer was freed fromexcess iminothiolane and any monomer by Z-250 chromatography and reactedwith a seven-fold molar excess of either crosslinker 3 or 4 over thefree SH concentration (approximately 1.5 SH/mol diphtheria toxinmonomer) for five minutes at pH 8.5. The solution was then diluted tentimes with water and made 40% in DMSO to dissociate uncrosslinkedspecies. The crosslinked dimer was isolated by chromatography on a Z-250column.

The same procedures were used to obtain crosslinked dimers of abrin andcrosslinked complexes of asialofetuin and ricin with crosslinker 1.

Proteins Which Do not Form non-covalent Aggregates

Anti-CD5-DT conjugate was crosslinked with crosslinker 1 A nicked DTmonomer, 10-15 mg/ml in 0.4 M Na borate, pH 8.0, was derivatized with0.5-1 mM iminothiolane for thirty minutes, and freed of residualiminothiolane by chromatography on G-25F in pH 8.5 buffer. Thederivatized diphtheria toxin was reacted with a 15-fold mol/mol excessof crosslinker 1 dissolved in dry dimethylformamide and after fiveminutes was rechromatographed on G-25F. The derivatized diphtheria toxinwas at that point a mixture of internally crosslinked diphtheria toxinand diphtheria toxin containing one free maleimide group. A 20-foldexcess of this diphtheria toxin mixture was then reacted with thiolatedT101 as described above at pH 8.5 for thirty minutes. The reactants andproducts were separated on Z-250 chromatography. The yield ofcrosslinked conjugate was from about 70 to about 90% by weight, based oninput antibody. Mono and bi-toxin conjugates were separated by Z-250chromatography.

Anti-CD5 ricin linked with crosslinker 1 was prepared by an essentiallysimilar procedure.

For control studies, similar ricin and diphtheria toxin conjugates werecrosslinked with the non-cleavable crosslinking reagentbis-maleimidohexane (BMH) by similar methodology. The yields andchromatographic patterns of the non-cleavable products were essentiallyidentical to the cleavable analogs and served as non-cleavable controls.

Hydrolysis of Acid Cleavable Conjugates Resulting in the Release of theIndividual Proteins

Crosslinked proteins were stored at pH 8.5 in 90/10/1 buffer at 4° C.Acid pulsing was achieved by adding an amount of buffer of higherbuffering capacity to achieve the desired lower pH at 25° C. Thereaction was stopped by injection into a Zorbax 450 size exclusion HPLCcolumn operated at pH 8 5 in 0.1% SDS. Monomeric and dimeric diphtheriatoxin and antibody diphtheria toxin and ricin are all separable by sizeusing a U.V. detector fed to an integrator. The loss of the crosslinkedspecies was quantitated by the appearance of the uncrosslinked species.

Below pH 6.3, acid pulsing of diphtheria toxin was performed in thepresence of SDS to prevent precipitation of the diphtheria toxin. FIG. 4shows the dissociation of crosslinked diphtheria toxin dimers usingcrosslinkers 3 and 4 into monomers. The log of the fraction of remainingdimer decreases linearly with time, indicating a simple first orderprocess. Acid induced dissociation has also been observed bychromatography in the absence of SDS using 90/10/1 buffer.

The degree of cleavage has been observed down to 0.05 of the crosslinkedspecies and is limited only by the degree of formation of non-cleavabledisulfide bonds. The acid induced dissociation of the immunotoxinT101-DT crosslinked with crosslinker 2 is shown in FIG. 5 over a 3 pHunit range. Within experimental error, the observed first order rateconstant of dissociation, k, is linearly related to the hydrogen ionconcentration. This linear dependence gives the widest possible changein dissociation rates at any two different pH values, and is, a desiredfeature in a crosslinker which uses a 2 pH unit change (from vascular toacidified vesicle compartment) to achieve a change from a relativelystable state to a labile state. Specific acid catalysis is responsiblefor the linear dependence of k on [H⁺ ] and is a characteristic ofacetal, ketal, and orthoester hydrolysis.

Table I shows a comparison of the half-time of crosslinked proteindissociation determined for four different acid cleavable crosslinkersat 25° C. Between 25° C. and 37° C., hydrolytic rates change by lessthan two-fold. The pH stability of the series isacetal>ketal>orthoester, and is in general agreement with the literatureof the parent compounds. These crosslinkers were synthesized to cover awide range of hydrolytic rates. Major variables promoting acid liabilityinclude stabilization of the carbonium ion intermediate by electrondonating groups and increasing acidity of the parent alcohol.

                  TABLE I                                                         ______________________________________                                        Comparison of Crosslinked Protein Dissociation                                Half-lives at pH 5.5                                                                             Acid               T.sub.1/2                               Cross-             Cleavable Reactive pH 5.5                                  linker                                                                              Type         Group     Groups   hours                                   ______________________________________                                        4     Homobifunctional                                                                           Orthoester                                                                              bis maleimide                                                                          0.1                                     3     Homobifunctional                                                                           Orthoester                                                                              bis maleimide                                                                          0.3                                     1     Homobifunctional                                                                           Ketal     bis maleimide                                                                          0.7                                     2     Hetero-      Acetal    maleimide,                                                                             139.                                          bifunctional           NHS ester                                        ______________________________________                                         half-lives determined at pH 6.5 ± 0.1 for 4, and 3, and extrapolated t     pH 5.5 assuming a linear dependence of k.sub.diss on [H.sup.+ ]. All          measurements at 25                                                       

Blocking and Unblocking of a Protein Toxin Functional Domain with theAid of an Acid Cleavable Crosslinker

Cold induced dimerization of diphtheria toxin has been shown to resultin a functional loss of the diphtheria toxin binding domain and acorresponding loss of toxicity (Carroll et al., Biochemistry25:2425-2430, 1986). Treatment of dimers with DMSO converts dimers tomonomers (unnicked) and restores binding and toxicity.

Unnicked diphtheria toxin dimer was isolated and crosslinked withcrosslinker 3, forming a covalently stable dimer which could not bedissociated with 40% DMSO. Over 90% of the crosslinked dimer toxicitywas lost as assayed by inhibition of protein synthesis on Vero cellsfollowing a thirty minute incubation, wash, and reincubation for 2.5hours, as shown in FIG. 6. The dimer was acid pulsed from pH 8.5 to pH6.5 for two hours and chromatographed in 90/10/1 buffer on Z-250.Approximately 1/2 of the dimer peak was converted to monomer. Both peakswere isolated. The dimer peak toxicity was unchanged, while the monomerpeak toxicity was increased. The unnicked diphtheria toxinderivatization with iminothiolane accounts for a 0.5 log loss ofdiphtheria toxin toxicity over untreated unnicked diphtheria toxinmonomer.

Enhanced toxicity of Immunotoxins Constructed with Ketal Based CleavableCrosslinker

The potency of Anti-CD5 immunotoxins constructed with the cleavablecrosslinker 1 is enhanced toward Jurkat target cells over thenon-cleavable crosslinker BMH. The potency increase is 1.75 logs or50-fold for the diphtheria based immunotoxin and 1 log for the ricinbased immunotoxin as shown in FIGS. 7a and 8a, respectively. The 50-foldenhancement for the diphtheria toxin conjugate is almost entirelyspecific for the antibody route, not being seen on the non-target Verocells (cf. FIG. 7b).

It is noted in FIG. 7b that upon conjugation with antibody using BMH,the diphtheria toxin potency is reduced 50-fold. Steric effects due toantibody conjugation could act to reduce toxicity at several steps alongthe intoxication pathway, including:

1) inhibition of binding to diphtheria toxin receptor;

2) inhibition of membrane translocation to the cytosol;

3) inhibition of diphtheria toxin receptor-mediated uptake to thecompartment productive of toxicity; and

4) various combinations of the above

It is expected that a cleavable conjugate would regain a potency losscaused by antibody induced steric inhibition of translocation. The gainin potency should occur on both target and non-target cellsAlternatively, a potency loss on non-target cells due to antibodyinduced steric constraints of diphtheria toxin binding should not beovercome by intracellular cleavage. Additionally, no increase in potencyon target cells would be expected if the entry route is largely antibodymediated, as is demonstrated by a 1 and 2.5 log shift to higherconcentrations by excess antibody for the bis-maleimidohexane (BMH) andthe cleavable conjugate, respectively. The cleavable conjugate does notrestore toxicity to non-target cells. However, when the entry route isvia the antibody on the target cells, the lost potency of the BMHconjugate is fully recovered by cleavable conjugate. This suggestseither that the diphtheria toxin receptor-mediated inhibited, or thatthere were various combinations of the above possibilities. When theuptake is via the antibody route, access to the compartment product ofdiphtheria toxin toxicity is apparently enhanced by cleavage of theantibody moiety. For the ricin conjugate, half of the 10-fold loss inpotency with the BMH conjugate is recovered on non-target cells and thefull 10-fold amount is recovered on target cells with crosslinker 1,indicating that a combination of steric factors are operating with thisconjugate.

The choice of BMH as a non-cleavable control crosslinker was dictated byits similar structure to crosslinker 3. BMH has six flexible methylenegroups separating the bis-maleimide moieties. Crosslinker 3 has sevenresidues (five methylene, two oxygen) in this position, and is somewhatless flexible due to the 120° oxygen bond angles and steric hindrancefrom the ketal methyl groups. BMH is the most flexible commerciallyavailable maleimide crosslinker. It has been noted that crosslinkershaving less flexibility than BMH, such asm-maleimidobenzoyl-N-hydroxysuccinimide ester and succinimidyl4-(p-maleimidophenyl)butyrate induce larger losses in toxicity of ricinand diphtheria toxin when conjugated to T101 as compared to BMH assayedon non-target cells.

Because of the high sensitivity of Jurkat cells to ricin, the ricinconjugate is partially inhibited by lactose. The entire toxicity is notbeing mediated by the antibody route. The diphtheria toxin conjugate islargely mediated by the antibody route since 100-fold excess of T101shifts the dose response curve by 2.5 logs to higher conjugateconcentrations.

Reversible Steric Inhibition of Toxin Receptors and Binding Domains byPolyethylene Glycol Conjugation via an Acid Cleavable Crosslinker

Mono methoxy polyethylene glycol of average molecular weight 5000 can beactivated with acylating groups capable of coupling to amino groups suchas NHS esters (Polysciences); phenyl-chloroformate (Veronese et al. App.Biochem. Biotechnol 11: 141-152, 1985) or 1,1'-carbonyldimidazole(Beauchamp et al., Anal. Biochem. 131: 25-33,, 1983). Activatedpolyethylene glycol was reacted with a twenty-fold molar excess ofcysteamine.2HCl at pH 8.5. The derivatized disulfide was then reducedwith 50 mM dithiothreitol and the polyethylene glycol-SH derivative wasfreed of excess reactants by size exclusion chromatography. Thepolyethylene glycol-SH was reacted with a fifteen-fold excess ofcrosslinker 1 at pH 8.0. The maleimide derivative was freed of excessreactant by size exclusion chromatography and was immediately reactedwith thiolated ricin or thiolated diphtheria toxin derivatized withiminothiolane to provide 4SH groups/mol of toxin. Coupling of thepolyethylene glycol-toxin to antibody is performed as described above orvia polyethylene glycol containing two maleimide residues by usingpolyethylene glycol containing from two to four OH groups and proceedingas above. Increased steric blockade can be provided by using highermolecular weight polyethylene glycol fractions such as PEG 20.

Multiple polyethylene glycol molecules have been coupled to diphtheriatoxin via crosslinker 1. Steric inhibition of toxicity to non-targetcells has been achieved (2.5 logs). Studies of hydrolytic rates ofrelease of free diphtheria toxin between pH 5.5 and 6.5 indicate thatthe dependency on [H+] is to a power greater than 1.

A Direct Demonstration that a Protein Conjugate Made with Crosslinker 1is Hydrolyzed within the Endosomal Compartment FollowingReceptor-mediated Endocytosis

The iron transporting protein diferric transferrin, Tfn, has been shownto undergo the following cyclic steps:

1) cell surface receptor binding;

2) receptor-mediated endocytosis;

3) acid promoted release of iron within the endosome following endosomalacidification;

4) exocytosis of receptor bound apotransferrin;

5) dissociation of apotransferrin to the medium.

(Klausner et al., JBC 258: 4715-4724, 1983; Ciechanover et al., JBC 258:9681-9689, 1983). The average transit time for steps 2-5 is about 15minutes.

The complex Tfn-S-[mal-x-mal]³⁵ S-cysteine where [mal-x-mal]representscrosslinker 1 was synthesized. Tfn was thiolated with iminothiolane,reacted with an excess of crosslinker I, and then reacted with ³⁵S-radiolabelled cysteine. A similar non-cleavable conjugate wasconstructed with BMH. The conjugates consisted largely of Tfn, butcontained some apoTfn. At approximately 77 nM, specific binding anduptake by 3×10⁶ K562 cells was 5.7% and 4.8% of the input tracers (2×10⁵CPM) after thirty minutes exposure for crosslinker 1 and BMH conjugates,respectively.

Following a loading period as described above conducted in the presenceand absence of energy inhibitors (50 MM deoxyglucose and 10 mM Naazide), the cells were washed, and fresh medium plus and minus energyinhibitors was added containing 30 microg/ml cold Tfn. The cells werereincubated for thirty minutes at 37° C. The supernatants wereharvested, made up to 0.5% in SDS and chromatographed by size exclusionon Z-450 column in 0.1% SDS in 0.1 M NaPi, (a mixture of mono- anddibasic sodium phosphate) pH 8.5, 1 mM EDTA. This chromatographicprocedure separates Tfn from apoTfn and both from free ³⁵S-cysteine-S-maleimide alcohol, the low molecular weight hydrolysisproduct of the crosslinker 1 conjugate.

The energy inhibitors stop the cycling of Tfn after one cycle, so thatsupernatants from energy inhibited cells contain Tfn and apoTfn loadedinto the cell and released from the surface membrane after at most onecycle through the cell. The supernatant from the non-inhibited cellscontains apoTfn loaded during the entire first incubation and exocytosedduring the second incubation, as well as the membrane boundcontribution. By subtracting the values of the inhibited cells from thenon-inhibited cell, one eliminates the membrane bound contribution andobtains the contribution of material which has undergone cycling throughthe endosome compartment of these cells.

FIG. 10 graphs the ³⁵ S-cysteine counts versus the retention time on thecolumn for both conjugates from the second incubation supernatants. Thenon-cleavable conjugate appears at the retention time of apoTfn,indicating that it has cycled through the cell and lost its iron. Lessthan 3% of the counts have been liberated by hydrolysis. In contrast,the conjugate constructed with crosslinker 1 has lost 65% of its ³⁵S-cysteine to a fraction eluting coincident with free ³⁵ S-cysteine (Thecolumn cannot resolve ³⁵ S-cysteine from the ³⁵ S-cysteine-maleimidealcohol hydrolytic product). The hydrolytic product has been largelyexocytosed to the medium, unlike the liberated iron. This presumablyreflects the lack of an accepter or receptor for the low molecularweight hydrolytic product in contrast to iron. Because the half-time ofhydrolysis of this cleavable conjugate at pH 5.5 is roughly equal to theTfn transit time (within a factor of 2), an appreciable fraction, 0.35,remains non-hydrolyzed.

In FIG. 9, the ricin binding site of an Anti-CD5 (T101) immunotoxin hasbeen reversibly blocked by the inclusion of asialofetuin linked to ricinvia crosslinker 1 A similar but non-reversibly blocked immunotoxin wasconstructed with BMH. On target cells, the reversibly blocked conjugateis 1 log more potent than the BMH conjugate. On non-target cells,toxicity of both are 2 logs less than ricin. On acid treatment, ricintoxicity is regained within 0.5 log of the original toxicity.

In FIG. 10, the intracellular hydrolysis of ³⁵ S-cysteine from theconjugate transferrin-crosslinker 1 ³⁵ S-cysteine is observed afterfollowing a 30 minute loading into K562 cells (2×10⁵ CPM) and a 320minute exocytosis period into fresh medium. The arrows indicate theelution times of apotransferrin, diferric transferrin, and cysteine onthe Z-450 column run in SDS. The graphed counts are the differencebetween the supernatant without energy inhibitors minus the supernatantwith energy inhibitors.

Coupling of multiple (2-4) molecules to diphtheria toxin via crosslinker1 has been performed. Steric inhibition of toxicity to non-target cellshas also been achieved (2.5 logs).

According to the present invention, proteins or toxins can be stericallyinhibited by using the acid cleavable crosslinkers of the presentinvention, so that the [H+]dependence is increased by a power greaterthan 1. Delays of hydrolysis of the conjugate are achieved at earlytimes at higher vascular pH values. This widens the therapeutic ratio ofan immunotoxin or reversibly blocked macromolecular prodrug.

The present invention is an improvement over the use of lactose to blockthe ricin binding outside the cell, as the ricin binding site isreversibly blocked by crosslinking with a non-covalent complex of ricinand asialofetuin (ASF). These complexes associate via the galactoseresidues on ASF and the ricin galactose binding site. Ricin and ASF arethiolated with 5 mM IT as previously described.m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) is added to T101,providing one derivatized maleimide per molecule of T101. ASF is addedat the rate of one mol per mol of T101. The T101-ASF complex is isolatedby chromatography on Z-250.

Thiolated ricin is added to the T101-ASF complex at 1 mol per mol ofantibody in the presence of borate buffer to reduce aggregation.Crosslinker 1 is added in excess, and crosslinks the remaining SH groupson the ASF to the ricin SH groups. The T101-ASF-ricin complex ispurified by chromatography on Z-250 in 90:10:1 buffer plus 100 mMlactose (used to dissociate non-crosslinked ricin). The successfulblockade of ricin galactose binding sites is demonstrated by the factthat the complex is not bound or retarded by affinity chromatographyover an ASF-sepharose column, in contrast to free ricin or T101-ricin.

For in vivo use, the exposed asialo-galactose groups on the ASF moietyof T101-ASF ricin are sialated using CMP:NANA-Galbeta(1,4)GlcNAC-alpha-2,6-sialyltransferase (Genzyme Corp.) according to thesupplier's instructions, except that the reaction pH is raised to pH8.0. This is done to prevent hepatic uptake of the immunotoxin via thehepatic asialoglycoprotein receptor.

FIG. 9, Panel B, shows that the T101-ASF-Ricin complex has a 100-foldreduction in toxicity (ricin route) on non-target cells. Acid treatmentreleases most of the ricin as judged by the 50-fold increase intoxicity. This fact was verified by HPLC. In panel A of FIG. 9, thetoxicity of T101-Ricin crosslinked with crosslinker 1 in the presence of100 mM lactose (normalized to ricin toxicity) is compared to theT101-ASF-Ricin without lactose. The ASF blocked ricin immunotoxincompares favorably with the immunotoxin utilizing lactose blockade. Thetoxicity of the ASF blocked immunotoxin is largely via the T101 route,as demonstrated by the loss of toxicity achieved by adding excesscompeting T101 (data not shown).

The data of FIG. 9 demonstrate that a functional ricin binding site isrequired within an intracellular compartment for full efficacy of theT101-ASF-ricin immunotoxin. This is demonstrated by the reduced efficacyof the immunotoxin made with the non-cleavable (BMH) crosslinker whichlacks the functional binding site. The blocked ricin binding site maythus be unblocked within the cell by using a crosslinker which iscleavable by an intracellular component, in this case, dilute acid.

As can be seen from the above, the data validate the concept ofreversible blockade of a toxin binding site in an immunotoxin to achieveenhanced efficacy while maintaining target cell specificity. Thisconcept can be applied to immunotoxins constructed with a wide varietyof toxins, monoclonal antibodies, and other targeting ligands. Althougha small class of targeting ligands which are rapidly internalized suchas transferrin and Anti-CD3 monoclonal antibodies and other targetingmoieties operating on receptors in very high numbers (over 0.5×10⁶ percell) may not require the toxin binding site for full immunotoxinefficiency, as disclosed by Greenfield et al. (1987) Science 238:635-639, but in general, most monoclonal antibody toxin-conjugatesrequire this feature for full efficacy.

Thus, the conjugates of the present invention can be effectively used tokill unwanted cell types in vivo and in vitro.

According to the present invention, the cellular component causingcleavage is not appreciably present in the serum, the cellular componentis present within a compartment to which the immunotoxin is routed bythe targeting moiety, and the intracellular cleavage is sufficientlyrapid to restore substantially full activity in which any cleavage ratein serum rate is at least 0.01 fold lower and preferably more.

Other examples of conjugates according to the present invention arelinkages consisting of peptide bonds or glycoside units which arecleavable by either intracellular cathepsins or glycosidases present ininternal vesicles and Golgi compartments, respectively. These linkagesare to be used in conjunction with agents which hinder toxin bindingsites, such as ASF or glycopeptides for ricin, and polyethylene glycolfor a variety of toxins as already described herein.

The conjugates of the present invention are particularly useful intreating AIDS, or acquired immune deficiency syndrome. These anti-humanpan T cell conjugates break the infectious cycle of AIDS by killing themajor cell types harboring HIV, T cells, and macrophages. For theT101-ASF-ricin immunotoxin, T cells are killed via the anti-CD5 moiety,and macrophages are killed via the ricin mannose residues which havehigh affinity for macrophages (cf. Cell 19: 207-215, 1980).Administration of the conjugates is preferably intraperitoneally orintravenously. Optimal dosages are a single administration of about 10to about 100 micrograms of ricin moiety/kg body weight.

The anti-CD5 moiety was chosen because the subset of T cell harboringvirus, CD4, is embraced by CD5. However, CD4 antigen density is known tosubstantially diminish following infection and viral replication, makingthis epitope unsatisfactory for targeting purposes. This therapy may beperformed in the presence of agents which inhibit viral replication.

Anti-CD5 immunotoxins made with mutant diphtheria toxins which areblocked in binding but not in translocation, such as CRM 107 and CRM103, or truncated toxin mutants, or chemically altered toxin havingsimilar properties, are also useful in AIDS therapy as described above,except that these immunotoxins lack macrophage and monocyte killingability. These functions must be provided by utilizing an immunotoxinmixture specific for all of the needed epitopes as was done in U.S. Pat.No. 4,520,226, which patent is incorporated herein by reference. Theimprovement herein is that each immunotoxin is constructed with acleavable crosslinker, providing increased efficacy.

The dosage of CRM 107 based immunotoxins ranges from about 300 to about300 micrograms/kg of body weight in terms of mutant diphtheria weightcontent, and from CRM 103 based conjugates, from about 3 to about 30micrograms/kg of body weight, administered one time only.

The crosslinked conjugates of the present invention may be used toprepare prodrugs, which can be used to deliver an amino-group-containingbiologically active substance to selected members of a heterogeneouspopulation of cells by exposing the cells to a complex formed bycrosslinking the active substance to a cell-binding partner specific fora cell-surface receptor of the selected cells. The compound bindsselectively to those cells, and the active substance is released fromthe complex by exposure to a pH sufficiently low to cleave thecrosslinker bond between the active substance and the crosslinker.

The conjugates of the present invention may be administered to a patientin a variety of forms, although intravenous or intraperitonealadministration of the active ingredient in a suitable pharmaceuticallyacceptable vehicle is the preferred delivery route.

Compositions within the scope of the present invention includecompositions wherein the active ingredient thereof is contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts, of course, is well within the skill in the art.

In addition to the conjugates of the present invention, thepharmaceutical compositions of the present invention may contain anysuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the conjugates intopreparations which can conveniently be used pharmaceutically.

Suitable formulations for parenteral administration include aqueoussolutions of the conjugates in water-soluble form. In addition,suspensions of the conjugates as appropriate oily injection suspensionsmay also be administered. Suitable lipophilic solvents or vehiclesinclude fatty oils, for example, sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension such as sodium carboxymethyl cellulose, sorbitol, and/ordextran. Optionally, the suspension may also contain stabilizers.

The heterobifunctional crosslinkers are superior to the homobifunctionalcrosslinkers for linking two different molecules. However, theheterobifunctional crosslinkers are more difficult to synthesize.

While the invention is described above in relation to certain specificembodiments, it will be understood that many variations are possible,and that alternative materials and reagents can be used withoutdeparting from the invention. In some cases such variations andsubstitutions may require some experimentation, but such will onlyinvolve routine testing.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept,and therefore such adaptations and modifications are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology herein is for the purpose of description and not oflimitation.

What is claimed is:
 1. A conjugate comprising a biologically activecomponent for inhibiting or killing unwanted cells crosslinked with acrosslinker to a targeting moiety comprising a binding partner to cellsurface features wherein the crosslinker can be cleaved under mildacidic conditions and wherein the crosslinker is formed from a compoundselected from the group consisting of compounds of the followingformulas: ##STR17## wherein n is at least 1; ##STR18## wherein n is atleast 1 and m is at least 1; ##STR19## wherein n is at least 1;##STR20## wherein n is at least 1; ##STR21## wherein n is at least 1, R'is selected from the group consisting of C₁ to C₈ alkyl groups and R isselected from the group consisting of H and C₁ to C₈ alkyl groups; and##STR22##
 2. The conjugate of claim 1, wherein the crosslinker is formedfrom the compound of the formula: ##STR23## wherein n is
 2. 3. Theconjugate of claim 1, wherein the crosslinker is formed from thecompound of the formula: ##STR24## wherein n is
 2. 4. The conjugate ofclaim 1, wherein the crosslinker is formed from the compound of theformula: ##STR25## wherein n and m are each
 2. 5. The conjugate of claim1, wherein the crosslinker is formed from a compound of the formula:##STR26## wherein n is
 2. 6. The conjugate of claim 1, wherein thetargeting moiety is an antibody.
 7. The conjugate of claim 1, whereinthe biologically active component is a cell toxin.
 8. The conjugate ofclaim 1, wherein the biologically active component is aribosomal-inactivating protein.
 9. The conjugate of claim 8, wherein thebiologically active component is selected from the group consisting ofricin, diphtheria toxin, diphtheria toxin mutants, abrin, melphalan,bleomycin, adriamycin, and daunomycin.
 10. The conjugate of claim 6,wherein the antibody is a monoclonal antibody.
 11. The conjugate ofclaim 1, wherein the biologically active component is selected from thegroup consisting of proteins, peptides, and nucleic acids.
 12. Theconjugate of claim 11, wherein the protein is an enzyme.
 13. Theconjugate of claim 1, wherein the biologically active component is adrug.
 14. The conjugate of claim 1, wherein the targeting moiety isselected from the group consisting of antibodies, cell membranetransport agents and protein hormones.
 15. A pharmaceutical compositioncomprising:a conjugate of a biologically active component crosslinkedwith a crosslinker to a targeting moiety comprising a binding partner tocell surface features wherein the crosslinker can be cleaved under mildacidic conditions and wherein the crosslinker is formed from a compoundselected from the group consisting of compounds of the followingformulas: ##STR27## wherein n is at least 1; ##STR28## wherein n is atleast 1 and m is at least 1; ##STR29## wherein n is at least 1;##STR30## wherein n is at least 1; ##STR31## wherein n is at least 1, R'is selected from the group consisting of C₁ to C₈ alkyl groups and R isselected from the group consisting of H and C₁ to C₈ alkyl groups;##STR32## a pharmaceutically acceptable carrier.
 16. The composition ofclaim 15, wherein the carrier is an isotonic solution.
 17. Thecomposition of claim 15, wherein the targeting moiety is an antibody.18. The composition of claim 15, wherein the biologically activecomponent is selected from the group consisting of proteins, peptides,and nucleic acids.
 19. The composition of claim 18, wherein the proteinis an enzyme.
 20. The composition of claim 15, wherein the biologicallyactive component is a drug.
 21. The composition of claim 15, wherein thetargeting moiety is selected from the group consisting of antibodies,cell membrane transport agents and protein hormones.